1. Recombinant Adenovirus In Molecular Biology & Medicine Will Herrick Peyton Group Meeting March 20, 2013

2. What Are Adenoviruses? Linear dsDNA virus 80-100 nm Nonenveloped Icosahedral structure: 1: penton capsomeres 2: hexon capsomeres 3: viral genomic DNA Capsomeres are the protein subunits that self-assemble into a capsid to enclose/protect viral DNA

3. Adenoviruses in Disease 57 human adenovirus serotypes Three major possible effects of infection: – Respiratory disease – Conjunctivitis – Gastroenteritis Enter cells via receptor- mediated endocytosis – i.e. cell membrane engulfs particle into vesicles called endosomes Infections: – Spreads primarily through respiratory droplets – Can cause severe respiratory problems including pneumonia and symptoms similar to whooping cough, strep throat Very rarely fatal – No vaccines, no therapies, NO TREATMENTS AVAILABLE AT ALL!

4. Adenovirus Infection & Replication Cell surface attachment – Involving integrins (alphaVs) and another receptor Endocytosis and shedding of capsid dsDNA entry into nucleus Host RNA polymerase makes viral proteins – Transcription factors – Viral DNA polymerase – Modify host gene expression i.e. block apoptosis Later, viral DNA polymerase and transcription factors make rest of viral proteins In nucleus, capsids assemble around viral DNA. Cell lyses and releases particles.

5. Adenovirus Uses in Medicine Popular vector for gene therapy 1999: healthy 18-year old dies during clinical trial of adenovirus gene therapy – Freak occurrence, basically – But it has greatly slowed work on gene therapy in the USA ever since – FDA put ‘brakes’ on research, became less popular to study This gave China, with less regulations, an opportunity.. Without the stigma, China basically copied US ideas. 2003: first adenovirus-based gene therapy approved in China – pAd-p53: injected directly into tumors to express p53, which suppresses tumor growth and increases chemoradiation sensitivity (head & neck cancers) – $10,000/month! Not approved in USA. Can’t kill metastasized cells. Under investigation to treat: – Malignant mesothelioma & many other cancers – Cardiovascular disease VEGF to promote reendothelialization with stent implantation SERCA to modulate contraction

6. Adenovirus Uses In Molecular Biology Used primarily to induce expression of a gene or genes of interest. But why pick adenovirus over lentivirus? – Higher efficiency – Higher gene expression – Higher titers

7. How To Make Recombinant Adenovirus with the AdEasy System (Bert Vogelstein Lab) Insert gene of interest into ‘shuttle’ vector with DNA subcloning Adenovirus plasmid with adenovirus type 5 genome and sequences necessary for replication in E. coli

8. Insert Gene of Interest into Adenoviral Plasmid Linearize shuttle vector (left) with PmeI digestion Co-transform BJ5183 E. coli with both plasmids BJ5183 E. coli encode for genes which enable robust homologous recombination

9. Digestion with PmeI allows the left and right arms of the shuttle vector to overlap with regions of the adenovirus vector, and the gene gets inserted by bacterial machinery Selection with kanamycin only permits survival of colonies containing the intact plasmid with the gene of interest

10. Recombinant Adenovirus Production Adenovirus plasmid with gene of interest is linearized by PacI digestion, then transfected into mammalian cells expressinh E1a and E1b adenovirus genes – Necessary for replication, absent from adenovirus plasmid – Typically, human embryonic kidney (HEK) 293’s are used

11. Purifying Viral Titers HEKs are made to express the adenovirus vector by chemical transfection Then adenovirus is collected and used to infect more flasks of HEKs Then more adenovirus collected, more HEKs infected. Repeat until titer is high, than purify. Purification: – Vogelstein method uses cesium chloride ultracentrifugation Hard to do unless you own an ultracentrifuge… – Instead, I used a kit that can be done in the hood and only takes 10 minutes to use.

12. Conclusions Adenovirus are relatively straightforward to make and use – Very high infection efficiency and gene expression makes them valuable to labs such as ours They are potentially dangerous and do not create stable cell lines – So we cannot use them effectively in experiments with much cell proliferation, as new cells won’t have the gene Questions?